HDA9, a member of the deacetylase family, plays a vital role in regulating plant flowering time through flowering integrator SOC1 and AGL24. However, it remains elusive how HDA9 interacts with SOC1 and AGL24 in flowering time control. Here, HDA9 was cloned in Brassica juncea and then its three active sites were separately replaced with Ala via overlap extension PCR. Thus, mutants of HDA9D172A, HDA9H174A and HDA9D261A were constructed and fused into the pGADT7 vector. The yeast one-hybrid assays indicated that HDA9 mutants remained the interactions with the promoters of SOC1 and AGL24. Furthermore, the aforementioned results were confirmed in the dual luciferase assays. Interestingly, the DNA-protein interactions were weakened significantly due to the mutation in the three active sites of HDA9. It suggested that flowering signal integrator SOC1 and AGL24 were regulated by the key amino acid residues of 172th, 174th and 261th in HDA9. Our results provide valuable information for the in-depth study of the biological function and molecular regulation of HDA9 in Brassica juncea flowering time control.